Developing a Microsatellite Polymerase Chain Reaction System for Small Yellow Croaker (Larimichthys polyactis) and Its Application in Parentage Assignment
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Sample Collection and DNA Extraction
2.2. Genome Sequencing and Assembly
2.3. Microsatellite Selection and Primer Design
2.4. Amplification and Validation of Microsatellite Markers
2.5. Establishing Multiplex PCR Conditions
2.6. Statistical Analysis and Parentage Assignment
3. Results
3.1. Genome Assembly
3.2. Microsatellite Selection
3.3. Marker Validation
3.4. Establishing Multiplex PCR System
3.5. Parentage Assignment
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Group | Breeding System | Generation | Sex | Number of Individuals |
---|---|---|---|---|
1 | Artificial insemination | Parents | Male | 3 |
Female | 3 | |||
Offspring | - | 48 | ||
2 | Group mating | Parents | - | 90 |
Offspring | - | 230 | ||
3 | Wild type (Jeju Island) | - | - | 16 |
Metrics | Contig Level | Chromosome Level |
---|---|---|
Number of Contigs | 400 | 24 chromosomes + 177 unknowns |
Residues | 700,560,536 | 675,620,547 + 24,959,989 |
Ave. length | 1,751,401.34 | 3,485,475.30 |
Min. length | 31,123 | 31,123 |
Max. length | 11,060,126 | 40,851,670 |
N50 | 4,969,043 | 28,972,644 |
N (%) | 0 (0.00) | 20,000 (0.00) |
GC (%) | 291,237,301 (41.58) | 291,237,301 (41.58) |
Name | Motif | Size Range | Sequence | Fluorescence |
---|---|---|---|---|
LaP_01 | (AAG)16 | 82–148 | (F) TCTGGTTGCAATTTACGCTGC | FAM |
(R) TCTCTCAAACTATCAGACACAAACC | ||||
LaP_05 | (TCT)17 | 110–143 | (F) GACAAGGACAGGCTCAGTCG | HEX |
(R) AGACTGGCTCAGTGATCAGC | ||||
LaP_32 | (TAA)24 | 282–342 | (F) CCTTTCTGAATTGGGCGTGG | TAMRA |
(R) CCACATCTCACTCTGTGAATTAACG | ||||
LaP_39 | (GTA)24 | 183–246 | (F) CGTCTGCCATGTCAATGTGC | FAM |
(R) AAGGCTTACATCGGGTTGGG | ||||
LaP_50 | (ATT)24 | 278–362 | (F) CCCCTGACATCTCATCCAGC | HEX |
(R) CTGTAGCTTAATTGCTTATTACTCTGC | ||||
LaP_52 | (AAT)21 | 200–230 | (F) TTAGGAGACCTGTTTGCCGG | HEX |
(R) CACCAAGGCTACAGTTTGGC | ||||
LaP_73 | (GAA)17 | 190–235 | (F) TCAGTGAAACCGAAGTGGGC | TAMRA |
(R) GTCCCTCTCGTTATTCTTCTTGC | ||||
LaP_89 | (ATA)21 | 276–348 | (F) TTGTCAGACAGTAGGCACGG | FAM |
(R) ATTCAAGCATGCCACAGTCG | ||||
LaP_96 | (TTA)15 | 120–162 | (F) TGCATATCATACGTCTCCCTCC | TAMRA |
(R) CAAACTTGGAACCCCATCCC |
Locus | LaP_01 | LaP_05 | LaP_32 | LaP_39 | LaP_50 | LaP_52 | LaP_73 | LaP_89 | LaP_96 | |
---|---|---|---|---|---|---|---|---|---|---|
NA | Mating 1 | 4 | 3 | 4 | 3 | 3 | 3 | 4 | 4 | 4 |
Mating 2 | 3 | 4 | 4 | 3 | 4 | 3 | 3 | 4 | 4 | |
Mating 3 | 4 | 2 | 3 | 3 | 4 | 4 | 2 | 4 | 3 | |
Wild | 12 | 9 | 9 | 9 | 8 | 9 | 11 | 9 | 8 | |
PIC | 0.802 | 0.734 | 0.884 | 0.810 | 0.805 | 0.764 | 0.762 | 0.837 | 0.826 | |
Null | −0.055 | −0.039 | 0.088 | 0.075 | −0.025 | 0.020 | 0.034 | −0.053 | 0.028 |
Analysis Type | Confidence Level (%) | Critical Delta | Assignment (%) |
---|---|---|---|
Mother alone | |||
Strict | 95.0 | 0.00 | 20 (21) |
Relaxed | 80.0 | 0.00 | 36 (75) |
Unassigned | - | - | 12 (25) |
Father alone | |||
Strict | 95.0 | 0.00 | 8 (17) |
Relaxed | 80.0 | 0.00 | 36 (75) |
Unassigned | - | - | 12 (25) |
Parent pair | |||
Strict | 95.0 | 3.77 | 48 (100) |
Relaxed | 80.0 | 0.00 | 48 (100) |
Unassigned | - | - | 0 (0) |
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Noh, E.-S.; Shin, E.-H.; Kong, H.-J.; Kim, Y.-O.; Ryu, Y.-W. Developing a Microsatellite Polymerase Chain Reaction System for Small Yellow Croaker (Larimichthys polyactis) and Its Application in Parentage Assignment. Biology 2024, 13, 710. https://doi.org/10.3390/biology13090710
Noh E-S, Shin E-H, Kong H-J, Kim Y-O, Ryu Y-W. Developing a Microsatellite Polymerase Chain Reaction System for Small Yellow Croaker (Larimichthys polyactis) and Its Application in Parentage Assignment. Biology. 2024; 13(9):710. https://doi.org/10.3390/biology13090710
Chicago/Turabian StyleNoh, Eun-Soo, Eun-Ha Shin, Hee-Jeong Kong, Young-Ok Kim, and Yong-Woon Ryu. 2024. "Developing a Microsatellite Polymerase Chain Reaction System for Small Yellow Croaker (Larimichthys polyactis) and Its Application in Parentage Assignment" Biology 13, no. 9: 710. https://doi.org/10.3390/biology13090710
APA StyleNoh, E. -S., Shin, E. -H., Kong, H. -J., Kim, Y. -O., & Ryu, Y. -W. (2024). Developing a Microsatellite Polymerase Chain Reaction System for Small Yellow Croaker (Larimichthys polyactis) and Its Application in Parentage Assignment. Biology, 13(9), 710. https://doi.org/10.3390/biology13090710